2026-10-15T03:00:07Z https://www.scidb.cn/oai

10.57760/sciencedb.42307 2026-09-20T10:55:30Z

2026-09-20 Semi-automated strain engineering and screening in the thermophilic fungus Myceliophthora thermophila for recombinant protein production doi:10.57760/sciencedb.42307 en Thermophilic filamentous fungi are widely used for cellulase production at elevated temperatures; however, efficient recombinant protein production in these organisms remains limited because construction and screening of large transformant populations remain difficult. Here, Myceliophthora thermophila was engineered into a thermophilic fungal chassis through combinatorial deletion of genes associated with proteolysis, vesicle trafficking, and secretion, resulting in a hyper-secretory strain. An optimized expression cassette coupled with a laccase reporter enabled activity-based identification of productive transformants. Iterative genome engineering combined withQPix-assisted colony picking enabled parallel recovery and screening of fungal transformants, with 552 colonies recovered after transformation and dilution plating and 96 transformants further evaluated by ABTS-based plate screening. Targeted integration at the MtLacA locus enabled identification of productive recombinants through loss of laccase activity, and transformants frequently carried multicopy integrations. Through iterative screening and strain improvement, extracellular protein production reached ~1.1g/L in shake-flask cultures and 5.9 g/L in a preliminary 5-L fermentation after 144 h using glucose as the carbon source. The chassis also supported secretion of recombinant fungal enzymes and porcine growth factors. Collectively, these results establish a practical semi automated workflow for recombinant protein production and strain engineering in thermophilic filamentous fungi. Myceliophthora thermophila; filamentous fungi; recombinant protein production; semi-automated screening; thermophilic fungal chassis; activity-based screening 刘倩 Qian Liu 田朝光 Chaoguang Tian 弓朝晖Chaohui Gong Xinyu Yang杨心语 Xiucai Zhao赵秀彩 Fufan Gou苟福凡 Jie Zhou周杰 Wenliang Sun孙文良 Tao Sun孙涛 RESTRICTED dataset Science Data Bank